A pan-cancer analysis of the role of USP5 in human cancers

Posttranslational modifications (PTM) such as acetylation, deubiquitination, and phosphorylation of proteins, play important roles in various kinds of cancer progression. Ubiquitin-specific proteinase 5 (USP5), a unique member of deubiquitinating enzymes (DUBs) which recognizes unanchored polyubiquitin specifically, could regulate the stability of many tumorigenesis-associated proteins to influence cancer initiation and progression. However, the diverse biological significance of USP5 in pan-cancer has not been systematically and comprehensively studied. Here, we explored the role of USP5 in pan-cancer using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) database, and we also acquired and analyzed data via various software and web platforms such as R, GEPIA2.0, HPA, TISIDB, cBioPortal, UALCAN, TIMER 2.0, CancerSEA and BioGRID. USP5 expression was high in most cancers and differed significantly in different molecular and immune subtypes of cancers. In addition, USP5 had certain diagnostic value in multiple cancers, and high expression of USP5 generally predicted poor prognosis for cancer patients. We also found that the most frequent genetic alterations type of USP5 was mutation, and the DNA methylation level of USP5 decreased in various cancers. Furthermore, USP5 expression correlated with cancer-associated fibroblasts (CAFs), endothelial cells (EC) and genetic markers of immunodulators in cancers. Moreover, the result from single cell sequencing showed that USP5 could regulate several tumor biological behaviors such as apoptosis, DNA damage and metastasis. Gene enrichment analysis indicated “spliceosome” and “RNA splicing” may be the critical mechanism for USP5 to involve in cancer. Taken together, our study elucidates the biological significance of USP5 in the diagnosis, prognosis and immune in human pan-cancer.

USP5 expression in molecular and immune subtypes of cancers. TISIDB database (http:// cis. hku. hk/ TISIDB/) which composed of many data types to evaluate the interaction between cancer and immune system was used to analyze the relationship between USP5 expression and molecular or immune subtypes in pancancer.
Diagnostic value analysis. The receiver operating characteristic (ROC) curve was used to estimate the diagnostic value of USP5 in pan-cancer, via using the data of the mRNA expression of USP5 in cancer and normal tissues in TCGA and GTEx. Package "pROC" (v1.17.0.1) was used to calculate the ROC curves, and the "ggplot2" (v3.3.3) package was used for plotting. The closer the area under the curve (AUC) is to 1, the better the diagnostic accuracy is. AUC in 0.5-0.7 means low accuracy, AUC in 0.7-0.9 means good accuracy, and AUC in 0.9-1 means high accuracy.
Genetic alteration and DNA methylation analysis. The cBioPortal (https:// www. cbiop ortal. org/) was searched for the gene alternations of USP5 in TCGA PanCancer Atlas Studies. The genetic alterations and mutation site information were explored with the "Oncoprint", "Cancer Type Summary" and "Mutations" modules. And the effect of the gene alterations of USP5 on clinical prognosis, including progress-free survival (PFS), DSS, disease-free survival (DFS), and OS, for all TCGA cancer types was analyzed in the "Comparison" module. Methylation level of USP5 in cancers and corresponding normal tissues was investigated in the UALCAN database (http:// ualcan. path. uab. edu/ analy sis. html).
Immunogenomic analyses. Various algorithms, such as EPIC, MCPCOUNTER, QUANTISEQ, TIDE, TIMER and XCELL were applied to analyze the relationship between USP5 expression and immune infiltration levels across all TCGA cancers, using TIMER2.0 tool. And we also investigated the correlations between USP5 expression and immunodulators, MHC molecules, chemokines, and chemokine receptors in pan-cancer from the TISIDB database.
Single cell sequencing. Using CancerSEA, we explored the correlation between USP5 expression and different functional status of cancer cells at the single cell levels. The threshold for correlation between USP5 and cancer functional status was set at correlation ≥ 0.3 and p value < 0.05. Expression profiles of USP5 at single cells was showed by the T-SNE diagrams.
Gene enrichment analysis. BioGRID was used to explore potential protein interactions with USP5.
GEPIA2.0 was applied to collect the top 100 USP5-correlated genes from all TCGA cancer and normal tissues. Then pairwise gene-gene Pearson correlation analysis was performed between USP5 and the selected genes. Heatmap was used to represent the expression status of selected genes containing the partial correlation (cor) and p value. Gene ontology (GO) and Kyoto encyclopedia of genes and genome (KEGG) enrichment analyses for USP5-correlated genes were conducted via the "clusterProfiler" (v3.14.3) and "org.Hs.eg.db" (v3.10.0) package 19 . "ggplot2" (3.3.3) was used to present the results. p value < 0.05 was considered to be statistically significant.

Results
USP5 expression in human organs/tissues and pan-cancer. The flowchart of our study was showed in Fig. 1. Initially, we examined the mRNA and protein expression levels of USP5 in various organs or tissues. The results obtained from consensus dataset created by combing the HPA and GTEx transcriptomics datasets showed that mRNA of USP5 mainly expressed in skeletal muscle, skin, tongue, parathyroid gland, testis, pons, esophagus, cerebral cortex, adrenal gland and thymus ( Supplementary Fig. 1A,B). In addition, USP5 is primarily    Fig. 2A). Moreover, the paired sample analysis showed that compared with paracancerous tissues, USP5 was overexpressed in BLCA, BRCA, CHOL, ESCA, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC and STAD. On the contrary, USP5 was decreased in KICH and PRAD. And no significantly differential expression of USP5 observed in COAD, PAAD, READ, THCA and UCEC (Fig. 2B). Given the lack of paracancerous tissues in some analysis, we further detected the expression differences of USP5 using the combination of TCGA and GTEx. And the results showed that low expression of USP5 was only observed in LAML, and high expression of USP5 was observed in ACC, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, KIRP, LGG, LIHC, LUAD, LUSC, OV, PAAD, PCPG, PRAD, READ, SKCM, STAD, TGCT, THCA, THYM, UCEC and UCS. There was no difference shown in BLCA, KICH and KIRC. Analysis of MESO, SARC and UVM was not possible due to lack of sufficient normal samples (Fig. 2C). Furthermore, we used GEPIA2.0 to explore the effect of USP5 mRNA expression on patient's pathological stages. And we found that the expression of USP5 was significantly correlated with the pathological stages of CESC, KIRC, LIHC, LUAD, OV and PAAD ( Fig. 2D). At last, we further explored the protein expression level of USP5 in pan-cancer using the National Cancer Institute's CPTAC dataset and the IHC results provided by the HPA dataset. The result of CPTAC analysis indicated that the protein expression of USP5 was up-regulated and correlated with pathological stages in clear cell RCC (renal cell carcinoma) and OV (Supplementary Fig. 2A,B). And the immunochemistry results of the Human protein atlas showed that staining intensity of USP5 was greater in many cancers, mainly including BRCA, LIHC, OV, PRAD, READ and UCEC, which was consistent with the analysis result of the mRNA expression level of USP5 from TCGA + GTEx (Fig. 3A-F). Overall, USP5 was overexpressed in most cancers.

USP5 expression in different molecular subtypes and immune subtypes of pan-cancer.
We analyzed the correlation between USP5 expression and molecular or immune subtypes in pan-cancer from the TISIDB database. The results indicated that USP5 was expressed differently in 10 of 17 cancers for molecular subtypes, which showed the most increased level in the LumB subtype of BRCA (5 subtypes), HN-SNV subtype of COAD (4 subtypes), ESCC subtype of ESCA (5 subtypes), Classical subtype of HNSC (4 subtypes), C2b subtype of KIRP (4 subtypes), G-CIMP-low subtype of LGG (6 subtypes), primitive subtype of LUSC (4 subtypes), proliferative subtype of OV (4 subtypes), Kinasesignaling subtype of PCPG (4 subtypes) and CN_HIGH subtype of UCEC (4 subtypes) ( Genetic alteration of USP5 in pan-cancer. To investigate the genetic mutations of USP5 in pan-cancer, we used cBioPortal online platform based on TCGA data. The highest frequency of USP5 alteration appeared in UCEC, UCS, OV, SKCM, TGCT and LGG. And mutation, amplification and deep deletion were the most common genetic alterations types of USP5 (Fig. 8A). In addition, we found 149 mutation sites with missense mutation as the main alteration type in USP5. For instance, a missense mutation within the ubiquitin carboxylterminal hydrolase (UCH) damain, P650L/S alteration was detected in one case of UCEC and two cases of SKCM (Fig. 8B). Then the correlation between the putative CNA of USP5 and its gene expression in pan-cancer was shown in Fig. 8C,D. Moreover, compared with the unaltered group, the gene alteration of AGAP10P, CHD4, VWF, NCAPD2, GPR162, LRRC23, PTPN6, ATN1, LAG3 and CD4 was more predominant in group with USP5 alteration (Fig. 8E). Last, we studied the effect of USP5 genetic alteration on the prognosis of patients in pan- www.nature.com/scientificreports/ cancer, and the result indicated that patients with USP5 alteration had poor progress-free survival in pan-cancer (Fig. 8F), but not overall survival, disease-free survival and disease-specific survival ( Supplementary Fig. 3A-C).  www.nature.com/scientificreports/ Analysis of the methylation level of USP5 in pan-cancer. DNA methylation has been proved to play an essential role in the occurrence and progression of cancers. Using UALCAN database, we compared the methylation level of USP5 between normal and cancer tissues. We found that the promoter methylation    www.nature.com/scientificreports/ Also, we observed that USP5 was correlated with most immune inhibitors and immune stimulators except for KIR2DL1, KIR2DL3 and TNFSF18 in pan-cancer ( Supplementary Fig. 5A,B). In terms of major histocompatibility complexes (MHCs), USP5 was positively associated with most major histocompatibility complexes in KIRC, KIRP and UVM, and negatively associated with most major histocompatibility complexes in ESCA, KICH, LUSC and TGCT (Supplementary Fig. 5C). Moreover, we found that USP5 showed certain correlation with majority of chemokines with the exception of CCL1, CCL16, CCL27, CCL24 and CCL25 in pan-cancer ( Supplementary  Fig. 5D). Meanwhile, a negative correlation between USP5 and most chemokine receptors could be found in the majority of malignant tumors especially in ESCA, KICH, LUSC and TGCT ( Supplementary Fig. 5E).

Functional states analysis of USP5 at single cell levels.
Using the CancerSEA, we investigated the functional states of USP5 at single cell levels in various cancers. The results indicated that USP5 was positively correlated with angiogenesis, differentiation, hypoxia, inflammation and metastasis, and negatively correlated with apoptosis, cell cycle, DNA damage, DNA repair, invasion, metastasis, quiescence and stemness (Fig. 11A). Then, the association between USP5 and specific cancer types was further examined. And we observed that USP5 showed positive correlation with hypoxia in LUAD; with metastasis in RCC; with differentiation, angiogenesis and inflammation in RB (Retinoblestoma). In contrast, USP5 negative correlated with DNA repair in ALL (Acute Lymphoblastic Leukemia); with stemness in PC (Prostate cancer); with cell cycle in CRC (Colorectal cancer); with DNA repair, cell cycle and DNA damage in RB; with DNA repair, DNA damage, apoptosis, invasion, metastasis and quiescence in UM (Uveal Melanoma) (Fig. 11B-H). Additionally, T-SNE diagrams were used to display USP5 expression profiles at single cell levels from ALL, LUAD, RCC, PC, CRC, RB and UM (Fig. 11I-O).
Functional enrichment analysis of USP5 in pan-cancer. Finally, we screened out the USP5 coexpressed genes for a series of pathway enrichment analyses to understand the molecular mechanism of the USP5 gene in carcinogenesis and development. We first collected 179 molecules that interacted with USP5 via the BioGRID web service (Fig. 12A). Then we used GEPIA to acquire the top 100 USP5 co-expressed genes in pan-cancer. Among these, MLF2, COPS7A, PEX5, DDX47, STRAP and MRPL51 displayed strong correlations with USP5 in most cancer types (Fig. 12B,C). Furthermore, GO and KEGG enrichment analyses were used to reveal that USP5 co-expressed genes play a critical role in the regulation of spliceosome, RNA splicing, catalytic activity acting on RNA and histone binding in tumor pathogenesis (Fig. 12D).

Discussion
Emerging studies indicated that USP5, a unique member of DUBs that can specifically recognize unanchored polyubiquitin, play an essential role in regulating the repair of DNA double-strand breaks 10 , inflammatory responses 11 , and stress responses 12 . Meanwhile, USP5 could regulate the stability of many tumorigenesis-associated proteins to influence the progression of various cancers such as, hepatocellular carcinoma 17 , pancreatic ductal adenocarcinoma 20,21 , and non-small cell lung cancer (NSCLC) 14,15 . However, the significance of USP5 in pan-cancer has not been explored until now. In the present study, using multiple bioinformatics approach, we first revealed the abnormal expression of USP5 in human cancers and its expression level in different molecular and immune subtypes of cancers, then we explored the diagnostic and prognostic values of USP5 in various cancers. In addition, we analyzed the gene mutations and methylation levels of USP5 in pan-cancer. Furthermore, the correlation between USP5 expression and the infiltration levels of immune cells and regulators was investigated, and the underlying functions of USP5 at single-cell levels was also identified. Finally, we implemented the functional enrichment analysis to recognize the potential mechanisms for USP5 to influence the pathogenesis of cancers.
Many studies revealed that USP5 was overexpressed and closely correlated with occurrence and progression of various cancers 18 . In line with their researches, our findings from TCGA and GTEx also demonstrated that the expression level of USP5 was significantly higher in most cancers compared with their regular counterparts. In addition, we found that there were meaningful correlations between USP5 expression level and the different molecular or immune subtypes of cancers, which suggested to us to get a deeper understanding of USP5's function in cancer by targeting specific molecular or immune subtypes.
Based on our results, USP5 had a certain diagnostic accuracy (AUC > 0.7) in 20 cancer types, especially in predicting BRCA, CHOL, DLBC, LGG, LUSC, PAAD and THYM (AUC > 0.9), indicating the potential clinical application value of USP5 as a reliable diagnostic biomarker. Meanwhile, as a unique member of deubiquitinating Thus, the combination of USP5 with these tumor-related biomarkers separately may significantly improve the diagnostic accuracy for the above cancers. Using cox proportional hazards model and Kaplan-Meier analysis, we found that USP5 was negatively correlated with patients' prognosis generally. Specifically, USP5 expression indicated poor overall survival in patients with LAML, LIHC, LUAD, MESO, SKCM and UVM. In addition, we further analyzed the association between the expression level of USP5 and disease-specific survival or progressfree interval of cancer patients, and we proved that on the whole, USP5 exhibited risk role in MESO and UVM for overall survival, disease-specific survival and progress-free interval, in LUAD and SKCM for overall survival and disease-specific survival and in COAD for disease-specific survival and progress-free interval. In addition to the previous reported negative association between USP5 and the progression of BRCA 13 , BLCA 27 , CRC 16 , GBM 22 , LIHC 17 , melanoma 28 , NSCLC 15,26 , OV 23 and PAAD 20 , our result first showed that USP5 may emerge as a novel biomarker for predicting the prognosis of ACC, LAML and MESO, especially MESO. These results suggested that USP5 had important diagnostic and prognostic implications in various cancers, and may serve as a therapeutic target for precision oncology. USP5 gene was located in 12p13.31. The mutation of USP5 had been reported to cause several tissue disorders in drosophila, including severe defects in the eye development 29,30 . And the active site (C335A) mutation was www.nature.com/scientificreports/ proved to prevent the deubiquitination activity of USP5 31 . However, the studies on the USP5 gene alteration in human cancers were still rare. Here, we observed that USP5 genetic alterations, including mutation and amplification could be found in various cancer types. And the frequency of CHD4, VWF, NCAPD2, GPR162, LRRC23, www.nature.com/scientificreports/ and DNA conformation, played an essential role in multiple types of tumorigenesis 32,33 . In this study, evidence indicated that DNA methylation level of USP5 was down-regulated in the majority of common malignancies, which was consistent with the elevation of USP5 expression. Further studies on the gene alteration of USP5 and the relationship between DNA methylation and USP5 expression in cancer are needed. Tumor immune microenvironment (TIME), an essential part of tumor microenvironment (TME), mainly composed of immune cells, played a critical role in cancer progression [34][35][36] . Identifying new targets for immunotherapy was important for improving clinical outcomes, and the impact of USP5 on the TIME was rarely explored so far. The infiltrating immune cells were closely correlated with tumor growth, metastasis and invasion 37,38 . For example, cancer-associated fibroblasts, tumor-activated fibroblasts, could promote tumor development by secreting various cytokines or metabolites and forming barrier by shaping external-cellular matrix to inhibit the function of drugs and immune cells 39,40 . In addition, the proliferation of tumor endothelial cells had a protective function of tumor cells by preventing the blood lymphocytes from leaking out of the blood vessels and transporting them to the tumor 41 . And various immune cells recruited by progressed tumors can affect tumor growth, invasion and pathological angiogenesis by promoting the secretion of cytokines and chemokines 42,43 . In this study, we conducted analysis to assess the impact of USP5 on immune infiltration. And the result obtained through a variety of immune deconvolution methods showed that USP5 was significantly associated with the infiltration of immune cells, including CAFs and EC in certain tumors. Meanwhile, certain correlations between USP5 and various immunoregulation-related genes were found in many cancer types. Generally, our study suggested the potential value of USP5 as an effective target for immunotherapy to enhance the health of cancer patients. More preclinical and clinical trials are needed to explore the relationship between USP5 expression and immune checkpoints.
There was no doubt that USP5 played an essential role in tumorigenesis, but the underlying mechanism still remains elusive. Single-cell transcriptome sequencing was the key technique to analyze the potential functions of molecules at single-cell levels 44 . Using CancerSEA, we found that USP5 significantly correlated with many biological behaviors of cancers such as apoptosis, cell cycle, DNA damage, metastasis and invasion in several cancer types at single cell levels. Additionally, via the functional enrichment analyses of USP5 co-expressed genes, we showed that "spliceosome" and "RNA splicing" may be the critical mechanism for USP5 to involve in pan-cancer. Previous studies had demonstrated that USP5 could regulate cancers by mediating epithelial-mesenchymal transition, such as, in LIHC by stabilizing Snail Family Transcriptional Repressor 2 17 , in NSCLC by stabilizing Catenin Beta 1 25 and in BRCA by stabilizing Hypoxia-inducible factor 2α 13 . In addition, it had been found that silencing of USP5 may increase apoptosis and DNA damage to suppress the progression of PAAD 45 . Moreover, USP5 could promote cell cycle progression by preventing HERC4-mediated polyubiquitination of c-Maf in multiple myeloma 46 , and the downregulation of USP5 lead to the cell cycle arrest in UCEC 47 . Aberrant RNA splicing was thought to be critical in tumorigenesis 48 . In GBM, USP5 isoform2 was closely associated with the aberrant expression of polypyrimidine tract-binding protein 1 (PTBP1), an RNA splicing factor in GBM, and the forced expression of USP5 isoform1 inhibited cell growth and migration in two GBM cell lines, implying an essential role of individual USP5 isoforms generated by alternative splicing in gliomagenesis 49 . The potential molecular mechanism of USP5 related to tumorigenesis and whether USP5 could be a target for cancer therapy still need more experimental exploration.
In summary, using comprehensive bioinformatics analysis methods, our study explored the expression levels, potential diagnostic and prognostic value, genetic mutation, protein methylation, immunomodulatory effects and relevant signaling pathways of USP5 in pan-cancer. The results indicate that USP5 is overexpressed and has certain diagnostic value in various cancer types. In addition, USP5 may be a potential prognostic and immunerelated biomarker for cancer patients. This study clarifies the role of USP5 in tumorigenesis from multiple perspectives, providing some bases for further research on the specific mechanisms of USP5 in the progression and treatment of cancers.